The human multidrug resistance protein 3 (MRP3) is an organic anion and multidrug extruding transporter. It confers multidrug resistance in human cancer cells by decreasing the intracellular concentration of drugs, which results in cancer treatment failure. With several other bacterial and eukaryotic transporters, MRP3 is a member of the C-branch of the ATP binding cassette transporter (ABC Transporter). The human MRP gene family has multiple members, including MRP1, MRP2, and several other homologous genes MRP3, MRP4, MRP5, MRP6, and MRP8. MRP3 has a molecular weight of about 190-200 kDa and is closest to MRP1, with 58% amino acid identity.
In normal human tissues, MRP3 is expressed mainly in the liver, adrenal gland, placenta, testis, intestine, pancreas, colon, gallbladder, and at a relatively lower level in kidney. Under normal physiological conditions MRP3 is highly expressed on the basolateral membrane of enterocytes and cholangiocytes. Hepatic expression of MRP3 was found to be induced in MRP2-deficient rats and, in Dubin Johnson Patients, variable levels of human MRP3 protein expression were observed in hepatocytes. Hepatic expression of MRP3 has also been linked to an elevated concentration of serum bilirubin or its glucuronides which suggests that these endogenous compounds may be involved in the induction of MRP3. Additional in vivo studies have indicated that MRP3 is an inducible transporter under cholestatic conditions. Further, phenobarbitol was reported to also induce the expression of MRP3 without affecting the levels of serum bilirubin. Functional analysis has revealed that MRP3 is involved in the cellular extrusion of organic anions it can also transport bile acid.
Expression of MRP3 was also reported in some human cell lines, including Caco-2 and HepG2, and over expression of MRP3 has been observed in some tumor cell lines that have acquired the multidrug resistance phenotype. MRP3 has also been reported to play a role in progression of adult acute myeloid leukemia. MRP3 has been identified as a novel tumor marker for glioblastoma multiforme (GBM) by serial analysis of gene expression (SAGE). MRP3 RNA transcripts have been shown to be highly expressed in GBM tissues as compared with normal brain tissues.
Antibody-based therapy has proved very effective in the detection or treatment of various cancers. For example, HERCEPTIN™ and RITUXAN™ have been used successfully to treat breast cancer and non-Hodgkin's lymphoma, respectively. HERCEPTIN™ is a recombinant DNA-derived humanized monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor receptor 2 (HER2) proto-oncogene. RITUXAN™ is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. Both these antibodies are produced in CHO cells.
The antigen-binding fragments (i.e., Fv fragments) are the smallest entities that consistently maintain the binding specificity of the whole antibody. Advances in antibody technology have greatly facilitated the genetic manipulation of antibody fragments. To improve stability, recombinant single-chain Fv (scFvs) have been engineered with two variable domains covalently joined by a flexible peptide linker. ScFv antibodies can have advantages over conventional antibodies, as they can be produced in large quantities and they are relatively small in size which allows them to penetrate into cells such as tumor cells more efficiently and homogenously. Their smaller size also facilitates more rapid systemic and normal tissue clearance. Recent advances in phage display technology have also permitted the production of scFvs with a higher affinity, bypassing the immune system and immunization.
Monoclonal antibodies (MAbs) detecting an internal MRP3 epitope have been developed, but nothing has been reported that can detect an external MRP3 epitope.
The present invention provides alternative methods of detecting or targeting cells expressing MRP3 that overcome the limitations of conventional methods as well as offer additional advantages that will be apparent from the detailed description below.